An unpleasant visitor? The challenge of
having a red cell antibody visit the lab
Dr. Aseem Kumar Tiwari
Talk - Organization
1. Expected Antibodies and ABO blood group system
2. Other blood group systems and unexpected antibodies
3. Detection of unexpected antibodies
4. Antibody identification and Interpretation
5. Rule of Three and Phenotyping
6. Magnitude of thse problem
7. Antibody Screen Negative – Change in algorithm
8. Antibody Screen Positive – Approach to Patient
Page  2
Expected Antibody
Page  3
Laboratory Work-up – ABO system
Expected Antibody (Regular Antibody/Typical Antibody)
Forward(cell)
Reverse(serum)
Interpretation
-A -B
AC BC
+ -
- +
A
-
+
+ -
B
+ +
- -
AB
-
+ +
O
Page  4
-
Slide(1900)/Tube(1960)/Micro-titer plate(1980)/Solid phase
adhesion/Column Agglutination Technique
Other Blood Group Systems
Other blood group typing is not done routinely –
‘immunogenicity’ is considerably less than ABO and Rh
Page  5
Unexpected Antibody –
Unpleasant Visitor
Page  6
Other Blood Group Systems and
Unexpected Antibody
Unexpected (Irregular Antibody/Atypical)
Irregular Antibody – Develops either because of
Transfusion/Pregnancy
Page  7
Unexpected Antibody –
Detection
Page  8
Antibody Detection/Screening - Screening
Cells
Each vial (donor) has been phenotyped for each antigen
18 antigens are required on at least one of the vials: D, C, E, c, e,
M, N, S, s, P1, Lea, Leb, K, k, Jka, Jkb, Fya, Fyb
 Single Vial (Reagent Pooled ‘O” cells)
 Two Cells (Selectogen)
 Three Cells (Surgiscreen)
Protocol
 Reagent Pooled ‘O” cells for donors
 Three Cells (Surgiscreen) for patients
Page  9
Antigram- An example
 An antigram (2 or 3 cells) will list the antigens present in each vial
 A reaction to one or more cells indicates the presence of an
unexpected antibody
Anti
gen
s
D
C
E
c
e
K
k
F
y
F
y
J
k
J
k
a
b
a
b
L
ea
L
e
S
s
M N
b
P
1
I
S
3
7
AHG
-
2+
I
+
+ 0 + + + 0 0 + + + 0 + + + + 0 + -
II
+
0 + + 0 0 + + 0 + 0 0 + 0 + + 0 + - -
Page  10
Principle of testing ‘Unknown’ with ‘Known’
0
Antigram
Page  11
If If an antibody is detected,
it must be identified!
Page  12
Antibody Panel - Identification
An antibody panel usually includes at least 10 panel cells:
Page  13
Antibody Panel - Identification
Page  14
Antibody Panel - Identification
Page  15
Antibody Panel - Identification
Group O red blood cells
Page  16
Antibody Panel - Identification
Each of the panel cells has been antigen typed (shown on antigram)
+ refers to the presence of the antigen
0 refers to the absence of the antigen
Example: Panel Cell #1 has 14 antigens present: C, c, e, f, M, N, S, s, P1, Lea, k, Fya,, Fyb and Jka
Page  17
Antibody Panel - Identification
The same phase (s) used in an antibody screen is used in a panel
• AHG
Page  18
AHG Phase - Identification
0
3
3
3
0
0
0
0
0
0
0 0
0
0 0
Page  19
Interpreting Antibody Panels
Few basic steps to follow when interpreting panels
1. “Ruling out” means crossing out antigens
that did not react
2. Circle the antigens that are not crossed out
3. Consider antibody’s usual reactivity
4. Look for a matching pattern
Page  20
1. Ruling Out
2+
0
0
2+
0
0
2+
0
2+
0
0
0
Page  21
0
0
0
0
0
0
0
0
0
0
0
0
Cross out antigens that show NO REACTION in any phase; do
NOT cross out heterozygous antigens that show dosage.
0
0
0
0
0
0
0
0
0
0
0
0












2. Circle antigens not crossed out
2+
0
0
2+
0
0
2+
0
2+
0
0
0
Page  22
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0












3. Consider antibody’s usual reactivity
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
0 0 0
Lea is normally a Cold-Reacting antibody (IgM), so it makes sense
that we see the reaction in the IS phase of testing; The E antigen will
Page  23
usually react at warmer temperatures












4. Look for a matching pattern
E doesn’t match and it’s a
warmer rx Ab
2+
0
0
2+
0
0
2+
0
2+
0
0
0
Page  24
…Yes, there is a matching pattern!
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0












Interpretation
antiLea
Page  25
Rule of three!
Rule of three must be met to confirm the presence of the antibody
Patient serum MUST be:
Positive with 3 cells with the antigen
Negative with 3 cells without the antigen
Page  26
Same example fulfills the “rule of three”
3 Positive
cells
3 Negative
cells
2+
0
0
2+
0
0
2+
0
2+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0












Panel Cells 1, 4, and 7 are positive for the antigen and gave a reaction at immediate spin
Page  27
Panel
Cells 8, 10, and 11 are negative for the antigen and did not give a reaction at immediate spin
Screening and Identification
only in AHG Phase (Customization !)
Page  28
Antibody Screen
Expected Antibody with 3 cell panel are
(c, k, kpb, Jsb, Leb, N and s)
Page  29
Antibody Identification
In 11 cell panel identified anti-”c” in the patient.
Page  30
Confirmed patient red blood cell with rare antisera ”c”
Antibody Panels - Interpretation
Few basic steps to follow when interpreting panels:
• “Ruling out” means crossing out antigens
that did not react
• Circle the antigens that are not crossed out
• Look for a matching pattern
Page  31
Another Example
Page  32
Another Example
Page  33
Phenotyping
Antigen typing the patient red cells can also confirm an antibody
If reagent antisera (of the suspected antibody)
is added to the patient RBCs, a negative
reaction should result…Why?
Individuals DO NOT make allo-antibodies
against antigens they have
Patient has NOT been recently transfused (donor cells could react)
Page  34
Magnitude of Problem
Page  35
Scale of Problem
40/32560 (0.12%) or approximately 1 in 1000
15 out of 2026 (0.75%)
18/531 (3.4%) or approximately 34 in 1000
(multi-transfused patients)
Rajendra Chaudhary, Nitin Agarwal.Safety of type and screen method compared
to conventional antiglobulin crossmatch procedures for compatibility testing in
Indian setting. 2011:5 (2):157-15
Page  36
Thakral B, Saluja K, Sharma RR, Marwaha N. Red cell alloimmunization in a
transfused patient population: a study from a tertiary care hospital in north
India. Hematology. 2008 Oct;13(5):313-8.
Rh System
14 anti-D (35%)
anti-D (5.6%),
6 anti-E (15%)
anti-E (22.2%)
3 anti-c (7.5%)
anti-c (38.8%)
2 anti-e (5%)
1 anti-C (2.5%)
3 anti-D with anti-C (7.5%)
Kidd System
1 anti-Jk(b) (2.5%)
anti-Jk(a) (5.6%)
Lewis System
1 anti-Le(a) (2.5%)
anti-Le(a) (11.1%)
1 anti-Le(b) (2.5%)
anti-Le(b) (5.6%)
5 anti-M (12.5%)
anti-M (11.1%)
MNS System
1 anti-s (2.5%)
INCONCLUSIVE
2 (5%)
Total
40/32560
Page  37
18/531
Thakral B, Saluja K, Sharma RR, Marwaha N. Red cell alloimmunization in a
transfused patient population: a study from a tertiary care hospital in north
India. Hematology. 2008 Oct;13(5):313-8.
Antibody Screen
Antibody
Screen
Screened
Negative
(999/1000)
Page  38
Screened
Positive
(1/1000)
Antibody Screen Negative
999/1000
Page  39
Type & Screen Vs Cross-match
45373 patients for whom a total of 61668 units of packed red blood cells
(PRBC) were cross-matched in the AHG phase using DiaMed; ID cards.
An antibody screen was carried out in all the patients using the DiaMed;
ID-DiaCell I+II+III. The AHG cross-match was also carried out for all
recipients, irrespective of the result of the antibody screen. The results
were compared to see if there were any cases where the antibody
screening was negative but the AHG cross-match showed
incompatibility.
Not a single case was found where the antibody screen was
negative and AHG cross-match showed incompatibility.
Type and screen policy can be safe, efficient, cost-effective, and
beneficial to the transfusion service in India.
Page  40
Pathak S, Chandrashekhar M, Wankhede GR. Type and screen policy in the blood
bank: Is AHG cross-match still required? A study at a multispecialty corporate
hospital in India. Asian J Transfus Sci 2011;5:153-6
Type & Screen Vs Cross-match
11000 patients were cross-matched in the AHG phase
Simultaneous Type and Screen.
Not a single case was found where the antibody screen was
negative and AHG cross-match showed incompatibility.
Dr. Dolly Daniel – Personal Communication
Page  41
Not as yet!
Evaluated safety of T & S procedure for PTT in comparison with
conventional test tube cross match.
Study did not show the T and S to reach the expected safety level of
99%, it has achieved a reasonable level of safety (91.6%).
Only one sample gave a negative antibody screen while
conventional crossmatch was incompatible. This may be due to a
rare antibody in the patient sera against which the corresponding
antigen was not present on the screening cells but present on the
donor red cell. A transfusion reaction would have occurred in this case
since it was reacting at 37΀C in AHG. Since, the screening panel used
in the present study was not from Indian subcontinent, therefore, it
would be advisable to prepare screening cell panels that include RBC
antigens which are prevalent within the local/regional population e.g, In,
Mi a etc.
Page  42
Rajendra Chaudhary, Nitin Agarwal. Safety of type and screen method compared
to conventional antiglobulin crossmatch procedures for compatibility testing in
Indian setting. 2011:5 (2):157-15
Can Cross-match be Omitted?
Over the 2-year interval of the study 9128 patients were entered. There
were 8936 patients (97.9%) with a negative antibody screen were
transfused a total of 10,899 red cell concentrates. Antiglobulin crossmatch
performed after the transfusion indicated that 168 red cell concentrates
(1.5%) would have been incompatible if the antiglobulin crossmatch had
been performed pretransfusion. 79.2% (103/130) were false positive
laboratory results. There were 27 transfusion episodes where the
antiglobulin crossmatch on blood transfused was positive due to an IgG
antibody. Even though these transfused red cell concentrates were
designated incompatible by the antiglobulin crossmatch, none of the
patients receiving this blood had clinical or serological evidence of
haemolysis. We concluded that the antiglobulin phase of the crossmatch
can be omitted from pretransfusion testing without putting patients at risk.
Page  43
Heddle NM, O'Hoski P, Singer J, McBride JA, Ali MA, Kelton JG. A prospective
study to determine the safety of omitting the antiglobulin crossmatch from
pretransfusion testing. Br J Haematol. 1992 Aug;81(4):579-84
Antibody Screen Positive
1/1000
Page  44
Antigen negative compatible blood
Antigen negative
Coomb’s Cross-match
Page  45
Antigen Negative Blood?
Antigen negative Blood
Random
Cross-match
Page  46
Type the
stocked blood
units
Rare Donor
Registry
Case study
Case 1
Plan of Action
Patient Name: Master S , (5
yr/M)
Repeat grouping
Hospitsl- RML hospital, New
Delhi
Screening with 3 cell panel
Identification with 11 cell panel
Problem: Grouping discrepancy
Blood group: forward- “A”
Reverse- “O”
Page  47
Finding ‘antigen negative blood
using rare antisera, if red cell blood
transfusion needed
Provisional diagnosis
Page  48
Findings…
Result of repeat blood grouping
=Forward- “A”, Reverse- “O”
Result of antibody detection(screening)
=Probability of Anti-M and Anti- Fyb
Result of antibody identification
= Anti- “M”
Confirmation of absence of M antigen on patients red cells
= No agglutination with Antisera-”M”
Page  49
Page  50
Conclusion:
Anti- “M” antibody present in the patient.
Out of 21 unit got only one “M” negative unit, preserved for Master S.
Page  51
Comparative study of frequency of Ag
Antigen
Pos. donor (%)
Neg. donor (%)
PGI Chandigarh
(Pos %)
PGI
Chandigarh
(Neg %)
D
85
93.4
6
6.59
93.39
6.61
C
77
84.60
14
15.38
84.76
15.24
c
57
62.63
34
37.36
52.82
47.18
E
23
25.27
68
74.79
17.9
82.1
e
88
96.79
3
3.29
98.3
1.7
K
4
4.39
87
95.6
5.56
94.44
k
91
100
0
0
100
0
Fya
79
86.81
12
13.18
86.75
13.25
Fyb
62
68.13
29
31.86
56.15
43.85
Jka
72
79.12
19
20.87
82.65
17.35
JKb
53
58.24
38
41.75
66.56
33.44
Lea
13
14.28
78
85.71
20.82
79.18
Leb
54
59.34
37
40.65
60.57
43.53
S
59
64.83
32
35.16
56.47
43.53
s
79
86.81
12
13.18
87.38
12.62
M
81
89.01
10
10.98
75.39
24.61
Page  52
N
53
58.24
38
41.75
61.51
38.49
Acknowledgements
Renee Wilkins, PhD, MLS(ASCP)cm CLS 325/435; School of Health
Related Professions; University of Mississippi Medical Center
Page  53
Thank You
Page  54
Antibody Crossm
screen
atch
Cause
Resolution
Pos
Neg
Antibody directed
against antigen on
screening cell
ID antibody, select
antigen negative
blood
Neg
Pos
Antibody directed
against antigen on
donor cell which may
not be on screening cell
OR donor unit may have
IgG previously attached
ID antibody, select
antigen negative
blood OR perform
DAT on donor unit
Pos
Pos
Antibodies directed
against both screening
and donor cells
Antibody ID, select
antigen negative
blood
Page  55